Thermodynamics of thermal and athermal denaturation of gamma-crystallins: changes in conformational stability upon glutathione reaction.
The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.
Kono
M
M
Eye Research Institute of the Retina Foundation, Boston, Massachusetts.
Sen
A C
AC
Chakrabarti
B
B
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003459:Crystallins; D005978:Glutathione / Q000494:pharmacology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D006358:Hot Temperature; D007908:Lens, Crystalline / Q000032:analysis; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D003459:Crystallins; D006146:Guanidines; D005978:Glutathione; D019791:Guanidine